Purification of a Recombinant Paraflagellar Rod Protein-5 from Trypanosoma cruzi

Author(s)

Kylie Howson

Faculty Mentor(s)

Gabrielle Stryker (Biological Sciences)

Abstract

Trypanosoma cruzi is a single-celled parasite found in the Americas, from the southern United States through South America, and is responsible for causing Chagas disease. This vector-borne parasite can infect all mammals, including humans, through exposure to the feces of Kissing bugs (Triatomine bugs). These large bugs feed on blood and can infest mud-walls or thatched roofs of rural homes. Trypanosomes contain a distinct structure known as the paraflagellar rod (PFR) that runs alongside the parasite’s flagellum. The PFR is required for cell motility and is a complex structure of cytoskeletal filaments in a lattice-like arrangement. The PFR constitutes an immunologically unique set of proteins that have been shown to protect against T. cruzi infection in mice. Paraflagellar rod protein-5 (PFR-5) is a putative minor component of the PFR of Trypanosomes that contains both PFR and SH3 protein domains. This research aims to identify the sub-cellular location of PFR-5 within the parasite T. cruzi. The 5’ end of the pfr5 gene has been subcloned into the PinPoint Xa expression plasmid in Escherichia coli (JM109) cells. My research has focused on induction, extraction, and purification of the soluble PFR-5 fragment. Protein purification is completed with pH manipulation of strep-avidin beads for protein collection and verified through SDS-PAGE and western blot. The goal is to immunize mice with the collected protein to generate PFR-5 specific antibodies for fluorescent microscopy. This research contributes to the understanding of the PFR in Trypanosomes and increases the number of potential vaccine target proteins for Chagas disease.

Keywords: Protein Purification, Trypanosoma cruzi, Paraflagellar rod

Presentation

12 thoughts on “Purification of a Recombinant Paraflagellar Rod Protein-5 from Trypanosoma cruzi”

  1. This was a really interesting project and I appreciate how you explained the various terms and lab work.

  2. Great presentation! What were some of the difficulties with protein purification and how did you troubleshoot them?

    1. Hi Paul, thank you for your question! One of the biggest challenges with protein purification was the beads/resin. Because they were extremely sensitive to pH, I had to ensure the pH was exactly where I needed it. There were a couple tricks that I found to be important; I calibrated the pH sensor often, used fresh NaOH and added it drop-wise.
      Another challenge occurred when beads would be lost from easy agitation in between addition of NaOH. After multiple attempts of troubleshooting, I found that a centrifuge could fix this problem, but only if it was turned off immediately after being started and allowed to stop spinning without the brake. There were other options that worked, but they were time extensive. The centrifuge allowed this process to be completed in less than two hours, where the other options easily took over five hours.

  3. Really compelling project; loved the graphics and your presentation of the material. What are your hopes for this work moving forward, especially given the disruption to your project due to the COVID19 pandemic?

    1. Thank you for your comments and question!
      At this point I hope another student continues this important research. As COVID vaccines are giving us hope to return to pre-pandemic life, students will have the ability to collect enough protein to immunize mice and locate PFR-5 bringing this project to completion. Not only will this add knowledge to the complex structure of the PFR but there is potential for future applications in vaccine development to prevent T. cruzi infection.
      As a student researcher, the process of protein purification gave me incredible knowledge and the ability to utilized what I learned at CWU. Regardless of my hopes for the completion of this project, I truly hope another student will benefit from this research as I have and expand their knowledge and skills as a scientist in the laboratory.

  4. Nice presentation Kylie! Glad to see you were able to get some protein purification, now the lab has a protocol to move forward with. Congrats on graduation as well :).

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